Department of Defense (Idea Award W81XWH-07-0406) - To Ulus Atasoy
NIH RO1 A1080870 - To Ulus Atasoy
NIH R21 A1079341 - To Ulus Atasoy
University of Missouri Institutional Funds - To Ulus Atasoy

Experiment preparation
Before starting experiment, it is critical to have all reagents, containers and utensils RNase free. Treat glassware with RNase inhibitor (RNaseZAP, Ambion) followed by rinsing with DEPC-treated water. Ensure that all reagents are confirmed as RNase free.
1. Prepare mRNP Lysate
<ol>
 <li>Grow and harvest exponentially growing tissue cells to produce between 2-5 mg of total protein for each RIP. 
 <ol class="lalpha">
 <li>Two P150 culture dishes are usually sufficient.</li>
 <li>For each RBP being investigated, total cellular number and protein amounts must be optimized to maximize appropriate target mRNA and RBP interactions. </li>
 <li>RIP for qRT-PCR analysis may require less cellular lysate (approximately 400 &mu;g total protein) due to its amplification and detection method especially for high abundance RBPs such as HuR, AUF1, TIAR.</li>
 </ol>
 </li>
 <li>Pellet cells via centrifugation at 200 x g for 8-10 min at 4 &deg;C, then wash three times with ice cold PBS.</li>
 <li>After final wash, gently flick the bottom of the tube and resuspend cell pellet in equal volumes of prepared polysome lysis buffer (PLB) with RNase and protease inhibitors. 
 <ol class="lalpha">
 <li>It's recommended to measure out the exact volume of cell pellet. </li>
 </ol>
 </li>
 <li>Gently pipet mixture (do not vortex) to break apart clumps of cells and incubate lysate on ice for 5 min. </li>
 <li>Centrifuge at 13,000 x g for 20 min at 4 &deg;C to clear the lysate of debris. </li>
 <li>Immediately transfer cleared supernatant to pre-chilled microfuge tube.</li>
 <li>Keep on ice and store at -80 &deg;C. 
 <ol class="lalpha">
 <li>Immediate freezing completes the proper lysis of the cells and prevents unwanted binding. Proceed with RIP immediately following sample thaw and keep samples on ice to avoid RNA degradation.</li>
 <li>This lysate can be stored for up to six months at -80 &deg;C. Avoid repeated freeze thaw cycles as this can lead to protein and/or mRNA degradation. </li>
 </ol>
 </li>
 <li>Quantitate protein concentration in lysate using standard Bradford Protein assay. </li>
</ol>
2. Coat Protein A Sepharose Beads with Antibody and Wash
<ol>
 <li>Pre-swell protein A Sepharose (PAS) beads overnight in NT2 buffer (3-4 volumes) with 5% BSA. Store at 4 &deg;C. 
 <ol class="lalpha">
 <li>Long term storage of up to several months at 4 &deg;C is possible when supplemented with 0.1% sodium azide.</li>
 <li>Protein Sepharose beads should be chosen based on isotype of target RBP antibody. Proteins A, G and A/G all have specific isotype targets and vary in target affinity. Protein A sepharose beads were used based on isotype specificity and affinity for HuR protein antibody.</li>
 </ol>
 </li>
 <li>Before use, remove excess NT2 buffer, so that final beads to buffer ratio is 1:1. </li>
 <li>Using 1.5 ml RNAse-free microcentrifuge tubes, remove 100 &mu;l of PAS slurry and add 30 &mu;g of antibody for each individual IP reaction (i.e., specific RBP and isotype control). </li>
 <li>Add 100-200 &mu;l of NT2 buffer to antibody bead mix. 
 <ol class="lalpha">
 <li>Mixture may be stored for several weeks at 4 &deg;C when supplemented with 0.1% sodium azide. </li>
 <li>An isotype-matched antibody or whole normal sera from same species should be used in parallel as an antibody control against background RNA. </li>
 </ol>
 </li>
 <li>Add appropriate antibody to bead mix and incubate overnight, tumbling end over end at 4 &deg;C. 
 <ol class="lalpha">
 <li>Optimize antibody titer for specific protein being investigated (1, 5, 10 or 30 &mu;g of antibody is usually sufficient).</li>
 </ol>
 </li>
 <li>Prepare antibody-coated beads immediately before use by washing with 1 ml of ice-cold NT2 buffer 5 times. 
 <ol class="lalpha">
 <li>Wash bead mix by centrifugation at 13,000 x g for 1-2 min at 4 &deg;C. </li>
 <li>Carefully remove the maximum amount of supernantant with hand pipettor or aspirator but be careful to avoid disrupting pellet.</li>
 <li>Washing helps remove unbound antibody as well as RNase contaminants from antibody mixture. </li>
 </ol>
 </li>
 <li>Once the final wash has been completed, resuspend the beads in 700 &mu;l of ice-cold NT2 buffer followed by treatment with various RNase inhibitors to protect the target mRNAs, including 10 &mu;l of RNase Out at 40 U/&mu;l, 10 &mu;l of 100 mM DTT and 15 &mu;l EDTA (15 mM). Bring volume to 1,000 &mu;l with NT2 buffer.
 <ol class="lalpha">
 <li>Vanadyl ribonucleoside complexes are not used due to inhibitory effect of EDTA. </li>
 </ol>
 </li>
</ol>
3. Immunoprecipitation and RNA Precipitation
<ol>
 <li>Optional Preclear Step: 
 <ol class="lalpha">
 <li>To reduce background, beads and control antibody may be used to pre-clear lysate. This may reduce signal in the output.</li>
 <li>This step may be necessary to reduce background when doing IP followed by microarray. It is generally not necessary for IP followed by qRT-PCR.</li>
 <li>Preclear with 15 &mu;g of isotype control for 30 min/4 &deg;C tumbling end over end. </li>
 <li>Add 50 &mu;l of preswollen PAS beads non-coated with antibody from step 2.1.</li>
 <li>Incubate 30 min/4 &deg;C with rotation end over end </li>
 <li>Centrifuge at 10,000 x g at 4 &deg;C. Save supernatant for IP. </li>
 </ol>
 </li>
 <li>Add 100 &mu;l of isolated cleared lysate (approximately 2-5 mg) to prepared antibody mix.
 <ol class="lalpha">
 <li>Diluting lysate will help to reduce background and nonspecific binding. </li>
 <li>Amount of lysate input may vary depending on detection method and abundance of RBP or efficiency of RBP antibody as noted in step 1.1.c. </li>
 </ol>
 </li>
 <li>(Optional) Immediately mix tube by gently flicking several times followed by brief centrifugation at 10,000 x g at 4 &deg;C to pellet beads and immediately remove 100 &mu;l of supernatant as a total input mRNA representation for qPCR analysis using standard RNA isolation techniques.
 <ol class="lalpha">
 <li>This step is to confirm that input lysate RNA is optimal for IP and should only be performed as a check step or as a troubleshooting step following RIP with poor RNA results. </li>
 </ol>
 </li>
 <li>Wrap tube in parafilm to ensure tight sealing and incubate at 4 &deg;C for 2 to 4 hr, tumbling end over end.
 <ol class="lalpha">
 <li>Timing on incubation should be optimized based on target abundance and should be minimized to avoid complex rearrangement or degradation. For some RBPs shorter incubations may be more optimal. </li>
 </ol>
 </li>
 <li>Pellet beads at 5,000 x g for 5 min at 4 &deg;C and save supernatant for potential analysis by Western blot. Store aliquots at -80 &deg;C.
 <ol class="lalpha">
 <li>Aliquots of supernatant with high amounts of residual target protein may indicate a failure of the protein to be precipitated by sepharose beads. </li>
 </ol>
 </li>
 <li>As described previously, wash beads 5 times with 1 ml of ice-cold NT2 buffer and centrifugation (5,000 x g for 5 min at 4 &deg;C) then remove supernatant with hand pipettor or an aspirator.
 <ol class="lalpha">
 <li>More stringent wash methods may be used in order to reduce background by supplementing NT2 buffer with sodium deoxycholate, urea or SDS. </li>
 <li>Keep samples on ice as much as possible and work quickly to minimize degradation of target mRNA. </li>
 </ol>
 </li>
 <li>Optional: Save a small aliquot (10%) of bead mix or run an additional sample in parallel for checking the IP efficiency of target protein using Western blot analysis. </li>
</ol>
4. DNase and Proteinase K Treatments
<ol>
 <li>After washing, resuspend beads with 100 &mu;l of NT2 buffer supplemented with 5 &mu;l of RNase-free DNase 1 (2 U/&mu;l).</li>
 <li>Keep at 37 &deg;C for 5-10 min. Add 1 ml of NT2 buffer and centrifuge at 5,000 x g for 1 min at room temperature.
 <ol class="lalpha">
 <li>A small (~10%) aliquot of beads may be used to check the IP efficiency by SDS-PAGE analysis. </li>
 </ol>
 </li>
 <li>Resuspend PAS pellet in 100 &mu;l NT2 buffer, 5 &mu;l Proteinase K (10 mg/ml), and 1 &mu;l 10% SDS. </li>
 <li>Incubate the resuspended bead mixture for 30 min at 55 &deg;C in a water bath, gently flicking at every 10 min.
 <ol class="lalpha">
 <li>Proteinase K will aid in the release of the RNP components.</li>
 </ol>
 </li>
 <li>Pellet beads at 5,000 x g for 5 min at room temperature (RT) and collect the supernatant (~100 &mu;l). </li>
 <li>Add 200 &mu;l NT2 buffer to beads, centrifuge 5,000 x g for 2 min at RT, collect supernatant (~200 &mu;l), and discard beads. </li>
 <li>Combine supernatants (100 &mu;l and 200 &mu;l) and add 300 &mu;l lower layer of acid phenol-CHCl3.</li>
 <li>Vortex, 1 min RT (or 37 &deg;C in shaker) and centrifuge at 16,000 x g at RT for 1 min.</li>
 <li>Collect 250 &mu;l of upper layer (DO NOT disrupt interface), add 25 &mu;l sodium acetate at pH 5.2, 625 &mu;l 100% ETOH and 5 &mu;l glycoblue, and mix well.</li>
 <li>Store overnight at -20 &deg;C.
 <ol class="lalpha">
 <li>To ensure proper recovery of RNA, the addition of glycoblue will increase the visibility of the RNA pellet by acting as a carrier molecule.</li>
 </ol>
 </li>
 <li>The following day, mix tubes by inversion 3-5 times, spin at 12,000 x g at 4 &deg;C for 30 min and discard the supernatant.</li>
 <li>Add 1 ml of 70% ETOH to the blue pellet and mix by inversion or vortexing.</li>
 <li>Centrifuge 12,000 x g at 4 &deg;C for 2 min.</li>
 <li>Discard supernatant and centrifuge 12,000 x g for 1 min at 4 &deg;C. </li>
 <li>Remove any residual 70% ETOH with a pipette and air dry pellet at RT for 5 min. </li>
 <li>Resuspend in 20-40 &mu;l of RNase/DNase-free water or another appropriate volume as needed.
 <ol class="lalpha">
 <li>Sample is now ready for further downstream applications such as qRT-PCR or microarray. </li>
 <li>Nanodrop spectrophotometry may be used in assessing sample concentration; however, with precious samples, it may be beneficial to avoid nanodropping samples as it can be wasteful and relatively inaccurate. We suggest normalizing samples to housekeeping gene transcripts to assess purity and IP efficiency for precious or low yield samples. </li>
 </ol>
 </li>
</ol>
5. Representative Results
If the procedure is optimized and performed correctly, the immunoprecipitation should yield significant enrichment of mRNA targets. Typically, depending on the RBP and its mRNA target(s), we see enrichment of approximately 10- to 50-fold when assessed by qRT-PCR. Many targets of RBPs can be discovered en masse using microarray analysis. However, this method is more sensitive to degradation as compared with qRT-PCR. Depending on the RBP, the number of targets and the efficiency of the reaction, microarray can reveal hundreds of novel targets, or it may only uncover a few, if any. For example, one of the better characterized RNA binding proteins HuR, post-transcriptionally regulates the expression and translation of many important physiological genes 1, 2. Isolation of the HuR-ribonucleo-complex via RIP-Chip in breast cancer cell lines, for instance, revealed enrichment of several important known HuR targets, including &beta;-actin using qRT-PCR as shown in Figure 1. In both cancer cell lines &beta;-actin is enriched 12- to 15-fold. Typically, when performed properly we see a significant enrichment of &beta;-actin in a variety of cell lines. However, if the RIP does not reveal any significant enrichment for &beta;-actin this indicates a problem with the RIP and the procedure may need to be repeated. Furthermore, microarray analysis of immunoprecipitated samples from these cell lines revealed distinct expression subsets of HuR targets in different estrogen receptor (ER) positive MCF-7 cancer cells versus ER negative MB-231 breast cancer cell lines as demonstrated in Figure 2 1. These targets fall into several categories: known and unknown HuR targets that were either associated or not associated with cancer. For example, CALM2 and CD9 are both cancer genes which were not previously identified as HuR targets. Using the microarray and confirming with qRT-PCR, CALM2 and CD9 were found to be 5- to 180-fold enriched in the HuR pellet indicating a prominent interaction between the HuR protein and these target genes.
<img src="/files/ftp_upload/3851/3851fig1.jpg" alt="Figure 1" /> 
 Figure 1. Immunoprecipitation and RIP in MB-231 (ER-) and MCF-7 (ER+) breast cancer cells. Immunoprecipitations were performed from MB-231 or MCF-7 cell lysates using anti-HuR monoclonal antibody (3A2) and IgG1 isotype control. A. IP Western of HuR revealed expected size band as detected by 3A2. Panel on right reveals amounts of HuR in input lysates used from both cell lines, with &beta;-tubulin as a loading control. B. Verification by quantitative RT-PCR showed fifteen- and eleven-fold enrichments of &beta;-actin, a known HuR target, in the 3A2 IPs from MB-231 and MCF-7, respectively. All &Delta;&Delta;CT values were normalized to GAPDH. Experiments were done in duplicate (n = 2).
 <img src="/files/ftp_upload/3851/3851fig2.jpg" alt="Figure 2" /> 
 Figure 2. HuR RIP-CHIP identifies distinct genetic profiles in ER+ and ER- breast cancer cells. HuR immunoprecipitations were performed from MB-231 or MCF-7 cell lysates using HuR antibody and IgG1 isotype control hybridized to Illumina Sentrix arrays (47,000 genes). Control signals were subtracted. Results represent cumulative data from 12 different arrays. Experiments were done in triplicate (n = 3) for each cell line with matching controls. Scales are log2.

<ol>
	<li>Calaluce, R., Gubin, M. M., Davis, J. W., Magee, J. D., Chen, J., Kuwano, Y., Gorospe, M., Atasoy, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+RNA+binding+protein+HuR+differentially+regulates+unique+subsets+of+mRNAs+in+estrogen+receptor+negative+and+estrogen+receptor+positive+breast+cancer.">The RNA binding protein HuR differentially regulates unique subsets of mRNAs in estrogen receptor negative and estrogen receptor positive breast cancer.</a> BMC Cancer. 10, 126-140 (2010).</li><li>Gubin, M. M., Calaluce, R., Davis, J. W., Magee, J. D., Strouse, C. S., Shaw, D. P., Ma, L., Brown, A., Hoffman, T., Rold, T. L., Ulus Atasoy, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+the+RNA+binding+protein+HuR+impairs+tumor+growth+in+triple+negative+breast+cancer+associated+with+deficient+angiogenesis.">Overexpression of the RNA binding protein HuR impairs tumor growth in triple negative breast cancer associated with deficient angiogenesis.</a> Cell Cycle. 9, 3337-3346 (2010).</li><li>Keene, J. D., Komisarow, J. M., Friedersdorf, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RIP-Chip:+the+isolation+and+identification+of+mRNAs,+microRNAs+and+protein+components+of+ribonucleoprotein+complexes+from+cell+extracts.">RIP-Chip: the isolation and identification of mRNAs, microRNAs and protein components of ribonucleoprotein complexes from cell extracts.</a> Nature Protocols. 1, 302-307 (2006).</li><li>Tenenbaum, S. A., Lager, P. J., Carson, C. C., Keene, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ribonomics:+identifying+mRNA+subsets+in+mRNP+complexes+using+antibodies+to+RNA-binding+proteins+and+genomic+arrays.">Ribonomics: identifying mRNA subsets in mRNP complexes using antibodies to RNA-binding proteins and genomic arrays.</a> Methods. 26, 191-198 (2002).</li><li>Tenenbaum, S. A., Carson, C. C., Atasoy, U., Keene, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+regulatory+analysis+using+en+masse+nuclear+run-ons+and+ribonomic+profiling+with+autoimmune+sera.">Genome-wide regulatory analysis using en masse nuclear run-ons and ribonomic profiling with autoimmune sera.</a> Gene. 317, 79-87 (2003).</li><li>Tenenbaum, S. A., Carson, C. C., Lager, P. J., Keene, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+mRNA+subsets+in+messenger+ribonucleoprotein+complexes+by+using+cDNA+arrays.">Identifying mRNA subsets in messenger ribonucleoprotein complexes by using cDNA arrays.</a> Proc. Natl. Acad. Sci. U.S.A. 97, 14085-14090 (2000).</li><li>Baroni, T. E., Chittur, S. V., George, A. D., Tennebaum, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+RIP-Chip+Analysis.">Advances in RIP-Chip Analysis.</a> Methods in Molecular Biology. 419, 93-108 (2008).</li><li>de Silanes Lopez, I., Zhan, M., Lal, A., Yang, X., Gorospe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+target+RNA+motif+for+RNA-binding+protein+HuR.">Identification of a target RNA motif for RNA-binding protein HuR.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 2987-2992 (2004).</li><li>de Silanes Lopez, I., Lal, A., Gorospe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HuR:+post-transcriptional+paths+to+malignancy.">HuR: post-transcriptional paths to malignancy.</a> RNA Biol. 2, 11-13 (2005).</li><li>Grigull, J., Mnaimneh, S., Pootoolal, J., Robinson, M. D., Hughes, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+mRNA+stability+using+transcription+inhibitors+and+microarrays+reveals+posttranscriptional+control+of+ribosome+biogenesis+factors.">Genome-wide analysis of mRNA stability using transcription inhibitors and microarrays reveals posttranscriptional control of ribosome biogenesis factors.</a> Mol. Cell Biol. 24, 5534-5547 (2004).</li><li>Hieronymus, H., Silver, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+RNA-protein+interactions+illustrates+specificity+of+the+mRNA+export+machinery.">Genome-wide analysis of RNA-protein interactions illustrates specificity of the mRNA export machinery.</a> Nat. Genet. 33, 155-161 (2003).</li><li>Hieronymus, H., Yu, M. C., Silver, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+mRNA+surveillance+is+coupled+to+mRNA+export.">Genome-wide mRNA surveillance is coupled to mRNA export.</a> Genes Dev. 18, 2652-2662 (2004).</li><li>Mukherjee, N., Corcoran, D. L., Nusbaum, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+Regulatory+Mapping+Indicates+that+the+RNA-Binding+Protein+HuR+Couples+Pre-mRNA+Processing+and+mRNA+Stability.">Integrative Regulatory Mapping Indicates that the RNA-Binding Protein HuR Couples Pre-mRNA Processing and mRNA Stability.</a> Molecular Cell. 43, 1-13 (2011).</li></ol>

The authors declare that they have no competing interests.

Due to the nature of this experiment, optimization and experience will be the only guaranteed ways to successfully acquire the intended results. In many steps of this procedure, temperature and efficient handling of the reagents and products are critically important. Proper planning and execution of technique will help insure that the experiment was performed in an appropriate timeframe at the optimal temperatures recommended. A major issue with RNA isolation experiments is the sensitivity of RNAs to degradation by RNases. All reagents need to be RNase free and stored or used in RNase-free containers. This is a critical step in ensuring the integrity of your mRNA sample. Even when the experiment is performed properly, however, the desired outcome may not be achieved due to the nature of the interaction between the RBP and its target mRNAs. 
One potential problem is having low or even no signal from the RNA isolated by RIP-Chip. Although there may be signal from total RNA, this may be the result of inadequate binding protein being pulled down by the beads. The first troubleshooting step is to confirm that the cellular lysate being used has adequate expression of the specific RBP. Upon confirmation, protein may be isolated after the final NT2 wash and resuspended in Laemmli buffer or another appropriate denaturing buffer and heated at 95 &deg;C for 5 min. Western blot analysis may be used on these samples in coordination with input lysate as well as negative controls to ensure sufficient pull down of associated protein.
Furthermore, because lysing the cell is required to access these components, the potential for abnormal and unwanted interactions between normally separated proteins and mRNA may be introduced. These interactions could potentially bind and "soak up" your target mRNAs or binding proteins through nonspecific interactions. Additionally, proteins in these varying conditions can fold in multiple variations and their binding motifs may become inaccessible to their target mRNAs, preventing their interactions. Both of these strengthen the importance of working efficiently as well as utilizing the optimal temperatures listed to limit these unwanted interactions. Additionally, optimization of washing conditions for each specific target protein will be critical to maximize the purity of the interaction. More stringent washing conditions may be needed. For example, the wash buffer may be supplemented with SDS or an appropriate amount of urea to reduce nonspecific interactions and background in your signal output. This will be completely dependent on the experimenter's target RBP as well as the target mRNA in their unique physiological conditions. Some conditions will not be suitable for certain mRNA analysis tools, which should be noted in preparation of samples.
Finally, though RIP is successful in the enrichment of RNA-RBP interactions, a well known issue with RIP (CHIP) method is the inability to identify the specific binding domains of the RBP on the transient mRNA targets. Several cross-linking techniques can be used followed by RIP to isolate unique sequence targets; however, the use of short-wave UV tends to lead to nucleic acid damage. A new method known as PAR-CLIP, or photoactivatable ribonucleoside crosslinking and immuoprecipitation, employs long-wave UV to incorporate thiouridine into nascent RNA allowing the identification of unique binding sites from both stable and transient RNA interactions.
Overall, RIP-Chip has been established as an excellent tool used to isolate and study the interactions between RNA binding proteins and their mRNA targets by our group as well as many other research groups. Though sensitive in nature and practice, proper execution of this procedure will yield the isolation of these RNP complexes, which until recently, have been inaccessible for discovery and analysis.

<table border="1">
<tbody>
 <tr>
 <td>Name of Reagent </td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>1 M dithiothreitol</td>
 <td>Fisher</td>
 <td>BP172-5</td>
 <td>DTT</td>
 </tr>
 <tr>
 <td>DNase 1</td>
 <td>Ambion</td>
 <td>2235</td>
 <td>RNase free</td>
 </tr>
 <tr>
 <td>Ethylendiamine Tetraccetic Acid</td>
 <td>Fisher</td>
 <td>BP118-500</td>
 <td>EDTA</td>
 </tr>
 <tr>
 <td>Glycogen</td>
 <td>Ambion</td>
 <td>9516</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1 HEPES</td>
 <td>Sigma</td>
 <td>H3375-100G</td>
 <td>pH 7.0</td>
 </tr>
 <tr>
 <td>Igepal Nonidet P-40</td>
 <td>USB</td>
 <td>78641</td>
 <td>NP40</td>
 </tr>
 <tr>
 <td>1 M KCl</td>
 <td>Fisher</td>
 <td>BP366-500</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1 M MgCl2</td>
 <td>Fisher</td>
 <td>BP214-500</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>NT2 Buffer</td>
 <td>*See Below</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polysome Lysis Buffer</td>
 <td>*See Below</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Protease inhibitor cocktail tablets</td>
 <td>Roche</td>
 <td>11873580001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Protein A Sepharose Beads</td>
 <td>Sigma</td>
 <td>P3391</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Proteinase K</td>
 <td>Fisher</td>
 <td>BP1700-100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RNase Out RNase inhibitor</td>
 <td>Invitrogen</td>
 <td>10777-019</td>
 <td>40 U/&mu;l</td>
 </tr>
 <tr>
 <td>1 M NaCl</td>
 <td>Fisher</td>
 <td>BP358-212</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Sodium dodecyl sulfate</td>
 <td>Fisher</td>
 <td>BP166-500</td>
 <td>SDS</td>
 </tr>
 <tr>
 <td>1 M Tris-HCl</td>
 <td>Fisher</td>
 <td>BP153-500</td>
 <td>pH 7.4</td>
 </tr>
 <tr>
 <td>Trizol</td>
 <td>Invitrogen</td>
 <td>15596-026</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vanadyl Ribonucleoside Complexes</td>
 <td>New England Labs</td>
 <td>S1402S</td>
 <td>VRC</td>
 </tr>
 </tbody>
</table>
 

<table border="1">
 <tbody>
 <tr>
 <td>Reagent Workup</td>
 </tr>
 <tr>
 <td>Prepare reagents in RNase/DNase-free, DEPC-treated glassware</td>
 </tr>
 <tr>
 <td>Polysome lysis Buffer</td>
 </tr>
 <tr>
 <td>100 mM KCl</td>
 </tr>
 <tr>
 <td>5 mM MgCl2 </td>
 </tr>
 <tr>
 <td>10 mM HEPES (pH 7.0)</td>
 </tr>
 <tr>
 <td>0.5% NP40</td>
 </tr>
 <tr>
 <td>1 mM DTT</td>
 </tr>
 <tr>
 <td>100 units/ml RNase Out</td>
 </tr>
 <tr>
 <td>400 &mu;M VRC</td>
 </tr>
 <tr>
 <td>Protease inhibitor cocktail tablet</td>
 </tr>
 <tr>
 <td>5 ml of Polysome lysis buffer</td>
 </tr>
 <tr>
 <td>Add 50 &mu;l of 1 M HEPES (pH 7.0)</td>
 </tr>
 <tr>
 <td>500 &mu;l of 1 M KCL</td>
 </tr>
 <tr>
 <td>25 &mu;l of 1 M MgCl2 </td>
 </tr>
 <tr>
 <td>25 &mu;l of NP40</td>
 </tr>
 <tr>
 <td>4.7 ml RNase-DNase-free H2O</td>
 </tr>
 <tr>
 <td>50 &mu;l of 1 M DTT</td>
 </tr>
 <tr>
 <td>12.5 &mu;l of 100 U/ml RNase Out</td>
 </tr>
 <tr>
 <td>200 &mu;l Protease inhibitor cocktail (dissolved according to manufacturer)</td>
 </tr>
 <tr>
 <td>10 &mu;l 200 mM VRC (at time of use)</td>
 </tr>
 <tr>
 <td>NT2 Buffer</td>
 </tr>
 <tr>
 <td>50 mM Tris-HCl (pH 7.4)</td>
 </tr>
 <tr>
 <td>150 mM NaCl</td>
 </tr>
 <tr>
 <td>1 mM MgCl2 </td>
 </tr>
 <tr>
 <td>0.05% NP40</td>
 </tr>
 <tr>
 <td>1 L of NT2 Buffer</td>
 </tr>
 <tr>
 <td>50 ml Tris (pH 7.4)</td>
 </tr>
 <tr>
 <td>30 ml 5 M NaCl</td>
 </tr>
 <tr>
 <td>1 ml 1 M MgCl2 </td>
 </tr>
 <tr>
 <td>500 &mu;l NP40</td>
 </tr>
 <tr>
 <td>820 ml RNase-DNase-free H2O </td>
 </tr>
 </tbody>
</table>

method for the isolation and identification of mrnas, micrornas and protein components of ribonucleoprotein complexes from cell extracts using rip-chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

Watch this Scientific Journal Video about Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip at JoV

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

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27.4K Views.  University of Missouri. A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

Video: Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Purification and Visualization of Influenza A Viral Ribonucleoprotein Complexes

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A nucleoprotein (NP) into viral ribonulceoprotein particles (vRNPs). The influenza vRNPs are enclosed within the viral envelope. During cell entry, however, these vRNP complexes are released into the cytoplasm, where they gain access to the host nuclear transport machinery. In order to study the nuclear import of influenza vRNPs and the replication of the influenza genome, it is useful to work with isolated vRNPs so that other components of the virus do not interfere with these processes. Here, we describe a procedure to purify these vRNPs from the influenza A virus. The procedure starts with the disruption of the influenza A virion with detergents in order to release the vRNP complexes from the enveloped virion. The vRNPs are then separated from the other components of the influenza A virion on a 33-70% discontinuous glycerol gradient by velocity sedimentation. The fractions obtained from the glycerol gradient are then analyzed on via SDS-PAGE after staining with Coomassie blue. The peak fractions containing NP are then pooled together and concentrated by centrifugation. After concentration, the integrity of the vRNPs is verified by visualization of the vRNPs by transmission electron microscopy after negative staining. The glycerol gradient purification is a modification of that from Kemler et al. (1994)1, and the negative staining has been performed by Wu et al. (2007).2

The genome of the influenza A virus consists of eight separate complexes of RNA and proteins, termed viral ribonucleoprotein complexes (vRNPs). This paper describes the glycerol gradient purification and transmission electron microscopy visualization of influenza A vRNPs.

The influenza A viral genome consists of eight negative-sense, single stranded RNA molecules, individually packed with multiple copies of the influenza A ...

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Isolation of Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing conditions could be achieved in different plant species. Native protein:protein and protein:nucleic acid complexes from phloem samples have as yet scarcely been analyzed, although they might play important roles in maintenance of this specialized compartment or in long-distance signaling. Large molecular assemblies can be isolated using a blue native gel electrophoresis (BN-PAGE). Their protein components can be separated by a subsequent sodium dodecyl sulfate PAGE (SDS-PAGE). However, proteins with similar molecular weights co-migrate, what can hinder protein identification by mass spectrometry. Combining BN-PAGE with two different denaturing gel electrophoresis steps, namely Tris-Tricine-urea and SDS-PAGE, enables the additional separation of proteins according to their hydrophilicity/hydrophobicity and thus increases resolution and the success of protein identification. It even allows distinguishing proteins that only differ in their posttranslational modifications. In addition, blue native northern blotting can be applied to identify the RNA components in macromolecular complexes. We show that our protocol is suitable to unravel the protein and RNA components of native protein:protein and ribonucleoprotein (RNP) complexes occurring in phloem samples. Combining a blue native PAGE with two different denaturing PAGE steps can help to separate different kinds of large protein complexes, and also enables an increased identification rate of their components by mass spectrometry. Furthermore, the protocol is robust enough to simultaneously detect potentially bound nucleic acids within single protein complexes.

Phloem Sap Sampling from Brassica napus for 3D-PAGE of Protein and Ribonucleoprotein Complexes

Here we present a protocol to analyze the protein composition of large native protein:protein and protein:nucleic acid complexes from oilseed rape (B. napus) phloem exudate using a 3D polyacrylamide gel electrophoresis (PAGE) approach combining blue native (BN) with two denaturing PAGEs followed by mass spectrometric identification.

Sampling the phloem of higher plants is often laborious and significantly dependent on the plant species. However, proteome studies under denaturing ...